Methionine, an essential amino acid for life, is used in many fields, including as a food additive, a feed, a material for various medicinal solutions and medicines, etc. Methionine acts as a precursor for choline (lecithin) and creatine and is used as a material for the systhesis of cysteine and taurine. Further, methionine plays a role in sulfur supply. S-adenosyl-methionine (SAM), derived from L-methionine, serves as a methyl donor in vivo and is involved in the synthesis of various neurotransmitters in the brain. Methionine and/or S-adenosyl-L-methionine (SAM) is/are also found to prevent lipid accumulation in the liver and arteries and to be effective for the treatment of depression, inflammation, liver diseases and muscle pain.
As summarized below, methionine and/or S-adenosyl-L-methionine has been found, thus far, to have in vivo functions of:
1) helping prevent lipid accumulation in the liver, where lipid metabolism is mediated, and in arteries and maintaining blood flow to the brain, the heart and the kidneys (J Hepatol. Jeon B R et al., March 2001; 34(3): 395-401).
2) aiding digestion, detoxifying and excreting harmful agents, and scavenging heavy metals such as lead.
3) acting as an excellent antidepressant when methionine is administered in a dose of 800-1,600 mg of methionine per day (Am J Clin Nutr. Mischoulon D. et al., November 2002; 76(5): 1158S-61S).
4) improving liver functions against liver diseases (FASEB J. Mato J M., January 2002; 16(1): 15-26). Particularly, attenuating alcohol-induced liver injury (Cochrane Database Syst Rev., Rambaldi A., 2001; (4): CD002235).
5) showing an anti-inflammation effect on osteoarthritis and promoting the healing of joints (ACP J Club. Sander O., January-February 2003; 138(1): 21, J Fam Pract., Soeken K L et al., May 2002; 51(5): 425-30).
6) acting as an essential nutrition to hair to prevent brittle hair and depilation (Audiol Neurootol., Lockwood D S et al., September-October 2000; 5(5): 263-266).
Methionine, useful in the food industry and the medicine industry, can be produced in a chemical route or a biological route.
On the whole, chemical synthesis for the production of L-methionine utilizes the hydrolysis of 5-(β-methylmercaptoethyl)-hydantoin. However, the chemical systhesis suffers from the problem of synthesizing methionine in a mixture of L- and D-forms.
In the biological route, advantage is taken of the proteins involved in methionine production. Biosynthesis of L-methionine is achieved by converting homoserine into methionine with the aid of enzymes encoded by metA, metB, metC, metE, and metH genes. In detail, homoserine acetyltransferase which is the first enzyme in a methionine biosynthesis pathway and encoded by metA functions to convert homoserine into O-succinyl-L-homoserine. Subsequently, O-succinyl-L-homoserine is converted into cystathionine by O-succinylhomoserine lyase which is encoded by metB. Cystathionine beta lyase which is encoded by metC is responsible for the conversion of cystathionine into L-homocystein. Two enzymes, cobalamin-independent methionine synthase and cobalamin-dependent methionine synthase, which are respectively encoded by metE and metH, can catalyze the conversion of L-homocysteine into L-methionine. At this time, the metF gene product, 5,10-methylenetetrahydrofolate reductase and the glyA gene product, serine hydroxymethytransferase, synthesize, in cooperation, N(5)-methyltetrahydrofolate that acts as the donor of a methyl group necessary for the synthesis of L-methionine.
In the biological route, L-methionine is synthesized through a series of organic reactions by the enzymes. Thus, these enzymes and proteins controlling them may be used in the genetic manipulation for improving and controlling L-methionine synthesis. For example, Japanese Pat. Laid-Open Publication No. 2000-139471 discloses an L-methionine production method using Escherichia sp. in which metBL is overexpressed in the presence of a leaky type of metK, with thrBc and metJ eliminated. US 2003/0092026 A1 describes a Corynerbacterium sp. that is modified to remove metD, a factor inhibitory to L-methionine synthesis, therefrom. US 2002/0049305 discloses that the production of L-methionine can be improved by increasing the expression of 5,10-methylenetetrahydrofolate reductase (metF).
These biological processes, even though having the advantage of producing only L-methionine, have low production capacity. Extensive attempts for the improvement in fermentological processes, nutritive medium compositions and chromatographic processes have been made to increase the productivity of L-amino acids including L-methionine, but are proven to be insufficient.
Furthermore, when methionine is synthesized in a certain level or higher, it, as the final product, inhibits through feedback the gene transcription of the starting protein metA, which initiates the biosynthesis of methionine. In vivo, the level of methionine is regulated in such a manner that the transcription of the metA gene is inhibited by methionine and the metA product is broken down by protease in a translation stage. Accordingly, methionine cannot be increased to a certain level in vivo solely by the overexpression of the metA gene (Dvora Biran, Eyal Gur, Leora Gollan and Eliora Z. Ron: Control of methionine biosynthesis in Escherichia coli by proteolysis: Molecular Microbiology (2000) 37(6), 1436-1443).
Leading to the present invention, intensive and thorough research on L-methionine production, conducted by the present inventors, aiming at overcomining the problems encountered in prior arts, resulted in the finding that on the basis of the fact that the metB gene product O-succinylhomoserine lyase is able to synthesize O-succinyl-L-homoserine, an intermediate of the methionine biosynthesis, from the threonine hydrolysate 2-oxobutanoate, in addition to having the function of converting O-succinyl-L-homoserine to cystathione (Markus C. Wahl et al., FEBS Letters, Volume 414, Issue 3, 15 September 1997, Pages 492-496, Hiroshi Kanzaki et al., FEMS Microbiology Letters, Volume 33, Issue 1, January 1986, Pages 65-68), L-methionine can be produced using a strain capable of producing a high level of L-threonine. Additionally, the present inventors overexpressed threonine dehydratase, O-succinylhomoserine lyase and cystathionine beta lyase in a non-methionine auxotroph derived from the strain capable of producing a high level of L-threonine. Consequently, it was possible to produce L-methionine at high yield, even in the presence of a high level of methionine without inhibition, as compared with the conventional method using the metA protein.
Further, co-expression of 5,10-methylenetetrahydrofolate reductase and serine hydroxymethytransferase, both involved in donating a methyl group in the methionine biosynthesis pathway, in a strain capable of producing a high level of L-threonine was found to make the strain independent of methionine for growth, and overexpression of glyA and metF genes in the non-methionine auxotroph derived from a strain capable of producing a high level of L-threonine allowed methionine to be produced at high yield.
Furthermore, the application of the L-methionine biosynthesis-involved genes and the methyl group donation-involved genes brought about higher yield in the production of methionine.